Hemostasis is the spontaneous process of stopping bleeding from damaged blood vessels. Precapillary vessels contract immediately when cut; within seconds, thrombocytes, or blood platelets, are bound to the exposed matrix of the injured vessel by a process called platelet adhesion. Platelets also stick to each other in a phenomenon known as platelet aggregation to form a platelet plug to stop bleeding quickly.
An intravascular thrombus results from a pathological disturbance of hemostasis. Platelet adhesion and aggregation are critical events in intravascular thrombosis. Activated under conditions of turbulent blood flow in diseased vessels or by the release of mediators from other circulating cells and damaged endothelial cells lining the vessel, platelets accumulate at a site of vessel injury and recruit further platelets into the developing thrombus. The thrombus can grow to sufficient size to block off arterial blood vessels. Thrombi can also form in areas of stasis or slow blood flow in veins. Venous thrombi can easily detach portions of themselves called emboli that travel through the circulatory system and can result in blockade of other vessels, such as pulmonary arteries. Thus, arterial thrombi cause serious disease by local blockade, whereas venous thrombi do so primarily by distant blockade, or embolization. These conditions include venous thrombosis, thrombophlebitis, arterial embolism, coronary and cerebral arterial thrombosis, unstable angina, myocardial infarction, stroke, cerebral embolism, kidney embolisms and pulmonary embolisms.
A number of converging pathways lead to platelet aggregation. Whatever the initial stimulus, the final common event is crosslinking of platelets by binding fibrinogen to a membrane binding site, glycoprotein IIb/IIIa (GPIIb/IIIa). Compounds that are antagonists for GPIIb/IIIa receptor complex have been shown to inhibit platelet aggregation (U.S. Pat. Nos. 6,037,343 and 6,040,317). Antibodies against GPIIb/IIIa have also been shown to have high antiplatelet efficacy (The EPIC investigators, New Engl. J. Med. (1994) 330:956-961). However, this class of antiplatelet agents sometimes causes bleeding problems.
Thrombin can produce platelet aggregation largely independently of other pathways but substantial quantities of thrombin are unlikely to be present without prior activation of platelets by other mechanisms. Thrombin inhibitors such as hirudin are highly effective antithrombotic agents. However, functioning as both antiplatelet and anti-coagulant agents, thrombin inhibitors again may produce excessive bleeding. (The TIMI 9a investigators, The GUSTO Iia investigators, Circulation, 90: 1624-1630 (1994); Circulation, 90: 1631-1637 (1994); Neuhaus K. L. et al., Circulation, 90: 1638-1642 (1994))
Various antiplatelet agents have been studied for many years as potential targets for inhibiting thrombus formation. Some agents such as aspirin and dipyridamole have come into use as prophylactic antithrombotic agents, and others have been the subjects of clinical investigations. To date, the powerful agents such as disintegrins, and the thienopyridines ticlopidine and clopidogrel have been shown to have substantial side effects, while agents such as aspirin have useful but limited effectiveness (Hass, et al., N. Engl. J. Med., 321:501-507 (1989); Weber, et al., Am. J. Cardiol. 66:1461-1468 (1990); Lekstrom and Bell, Medicine 70:161-177 (1991)). In particular, use of the thienopyridines in antiplatelet therapy has been shown to increase the incidence of potentially life threatening thrombotic thrombocytopenic purpura (Bennett, C. L. et al. N. Engl. J. Med, (2000) 342: 1771-1777). Aspirin, which has a beneficial effect on platelet aggregation (Br. Med. J. (1994) 308: 81-106; 159-168), acts by inducing blockade of prostaglandin synthesis. Aspirin has no effect on ADP-induced platelet aggregation, and thus has limited effectiveness on platelet aggregation. Furthermore, its well documented high incidence of gastric side effects limits its usefulness in many patients. Clinical efficacy of some newer drugs, such as ReoPro (7E3), is impressive, but recent trials have found that these approaches are associated with an increased risk of major bleeding, sometimes necessitating blood transfusion (New Engl. J. Med. (1994) 330:956-961). Thus it appears that the ideal “benefit/risk” ratio has not been achieved.
Recent studies have suggested that adenosine 5′-diphosphate (ADP), a common agonist, plays a key role in the initiation and progression of arterial thrombus formation (Bernat, et al., Thromb. Haemostas. (1993) 70:812-826); Maffrand, et al., Thromb. Haemostas. (1988) 59:225-230; Herbert, et al., Arterioscl. Thromb. (1993) 13:1171-1179). ADP induces platelet aggregation, shape change, secretion, influx and intracellular mobilization of Ca+2, and inhibition of adenylyl cyclase. Binding of ADP to platelet receptors is required for elicitation of the ADP-induced platelet responses. There are at least three P2 receptors expressed in human platelets: a cation channel receptor P2X1, a G protein-coupled receptor P2Y1, and a G protein-coupled receptor P2Y12 (also referred to as P2Yac and P2T). The P2X1 receptor is responsible for rapid calcium influx and is activated by ATP and by ADP. However, its direct role in the process of platelet aggregation is unclear. The P2Y1 receptor is responsible for calcium mobilization, shape change and the initiation of aggregation. P2Y12 receptor is responsible for inhibition of adenylyl cyclase and is required for full aggregation. (Hourani, et al., The Platelet ADP Receptors Meeting, La Thuile, Italy, Mar. 29-31, 2000)
Ingall et al. (J. Med. Chem. 42: 213-220, (1999)) describe a dose-related inhibition of ADP-induced platelet aggregation by analogues of adenosine triphosphate (ATP), which is a weak, nonselective but competitive P2Y12 receptor antagonist. Zamecnik (U.S. Pat. No. 5,049,550) discloses a method for inhibiting platelet aggregation in a mammal by administering to said mammal a diadenosine tetraphosphate compound of App(CH2)ppA or its analogs. Kim et al. (U.S. Pat. No. 5,681,823) disclose P1, P4-dithio-P2, P3-monochloromethylene 5′,5′″ diadenosine P1, P4-tetraphosphate as an antithrombotic agent. The thienopyridines ticlopidine and clopidogrel, which are metabolized to antagonists of the platelet P2Y12 receptor, are shown to inhibit platelet function in vivo (Quinn and Fitzgerald, Circulation 100:1667-1672 (1999); Geiger, et al., Arterioscler. Thromb. Vasc. Biol. 19:2007-2011 (1999)).
There is a need in the area of cardiovascular and cerebrovascular therapeutics for an agent that can be used in the prevention and treatment of thrombi, with minimal side effects, such as unwanted prolongation of bleeding, while preventing or treating target thrombi.